A. Field of the Invention
The present invention encompasses a method for introducing foreign genetic material into somatic and germ cells of the chicken and other avian species. The invention further encompasses a method for introducing foreign genetic material into a chicken embryo resulting in the production of a transgenic chicken.
B. Description of the Art
There has been much interest in introducing foreign DNA into eukaryotic cells. One reason for this interest is that some genetically caused diseases may be curable by introducing the foreign DNA into the cells, allowing the foreign DNA to express a protein that the genetically defective cell cannot express. Another reason for this interest is that certain eukaryotic cells may prove to be the most suitable hosts for the production of certain eukaryotic proteins. The feasibility of introducing foreign DNA into animal genomes and thus being able to alter the phenotype of an intact animal by the insertion of this foreign DNA has stimulated considerable interest in the development of methods for gene transfer.
Successful methods of transfer of foreign genes into the germ cells of chickens would permit basic studies of gene expression in the avian system, and ultimately would permit the introduction of genes that may be used for poultry improvement. Gene transfer into chickens could thus potentially affect the long-range improvement of poultry by giving the breeder a new tool--a molecular genetic approach to breeding problems. Several advantages of such an approach are that: (a) gene transfer provides a means of increasing genetic variation by the introduction of genetic material into the genome, which permits gene flow between vastly different organisms and which transcends the limits of sexual reproduction; (b) gene transfer permits the formation of new phenotypes which may have increased economic value; and (c) gene insertion may permit the transfer of favorable traits between various stocks of chickens without concomitant transfer of less favorable genes that occurs using current methods of backcrossing.
The ability to manipulate the mouse embryo has led to a variety of successful approaches to gene transfer in this species. These include microinjection of DNA (Palmiter and Brinster, Cell 41:343-345 (1985) retroviral infection of young embryos (Jaenisch, Proc. Natl. Acad. Sci. USA 73:1260-1264 (1976); Soriano et al., Science 234:1409-1413 (1986)), and genetic modification of cultured pluripotential embryonic stem cells which can contribute to the germ-cell lineage of chimeric mice. Bradley et al., Nature 309:255-256 (1984); Robertson et al., Nature 323:445-448 (1986). In particular, the production of transgenic mice is now routinely achieved by microinjection of foreign DNA into male pronuclei of fertilized eggs. Palmiter et al., Nature 300:611-15 (1982). Microinjected eggs are then implanted into the oviducts of one-day pseudopregnant foster mothers and carried to term. The newborn mice are then tested for the presence of the microinjected DNA by means known in the art and appropriate to detect the presence of the microinjected DNA. A manual of procedures for microinjection of foreign DNA into isolated mouse egg cells has been published. Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory (1986). More recently, similar microinjection methods have been successfully employed using rabbit, sheep and pig egg cells. Hammer et al., Nature 315:680-683 (1985).
In contrast, similar egg cell microinjection methods for gene transfer have not been successfully applied to chickens because of difficulties involved in manipulating the chicken embryo. When oviposition occurs, the embryo has already reached a stage corresponding to a mammalian late blastula or early gastrula, (Kochav et al., Dev. Biol. 79:296-308 (1980); Eyal-Giladi and Kochav, Develop. Biol. 49:321-337 (1976). Genetic manipulation of the embryo during earlier development requires reintroduction to the female or in vitro culture. Rowlett and Simkiss, Br. Poult. Sci. 28:91-101 (1987); Perry, Nature 331:70-72 (1987). Both are difficult procedures, whose efficiency may limit their application as a method of gene transfer.
Because morphological and developmental characteristics of the early chicken embryo and other avian embryos restrict the use of the microinjection methods of gene transfer that have been used so successfully in the mouse and other species, an alternative method for introducing genes into chicken embryos has been used which involves the infection of embryos with infectious retroviral vectors. Transfer of additional growth hormone genes into chicken somatic cells via a replication competent Rous sarcoma virus (RSV) vector (Souza et al., J. Exp. Zool. 232: 465-473), transfer of replication competent reticuloendotheliosis virus (REV) by injection of virus into developing ovarian follicles near ovulation (Shuman and Shoffner, Poult. Sci. 65: 1437-44 (1986)). and more recently, transfer of RSV derivatives into chicken germ cells (Salter et al., Poult. Sci. 65: 1445-1458 (1986); Salter et al., Virology 157: 236-240 (1987); Salter and Crittenden, Poult. Sci. 66: 170 (1987)) has been accomplished by infection of day-old chicken embryos with replication competent retroviral vectors. Sorge and Hughes, J. Mol. Appl. Genet. 1: 547-559 (1982); Hughes et al., Poult. Sci. 65: 1459-1467 (1986). This approach to avian gene transfer has advantages over DNA microinjection since the early chicken zygote is difficult to manipulate, and even a freshly laid egg contains thousands of cells (Eyal-Giladi and Kochav, supra; Kochav et al., supra). However, the use of replication competent vectors in gene transfer has significant disadvantages. Replication competent vectors result in gene transfer to susceptible cells at various stages of differentiation long after initial infection of the embryo, because they are capable of subsequent rounds of infection after introduction. This can make it difficult to determine the stage of development at which gene insertion takes place, or the cell lineage relationship within fully differentiated tissues. Furthermore, replication competent vectors also increase the potential for disease states associated with chronic viral infection. Salter, et al., Poult. Sci. 65:1445-1458 (1986). More importantly, it is unacceptable to introduce infectious replication competent retroviral vectors into the germ cells of poultry intended for commercial use. Therefore, there was a need for the development of efficient vectors and efficient procedures using such vectors that would permit insertion of a foreign gene into cells in an initial round of infection but wherein the vectors would not permit reinfection of the cells after the initial round of infection. Several replication-defective retroviral vector systems have been derived (Bosselman et al., Mol. Cell. Biol. 7:1797-1806 (1987); Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Mann et al., Cell 33:153-159 (1983); Miller and Buttimore, Mol. Cell. Biol. 6:2895-2902 (1986); Watanabe and Temin, Mol. Cell. Biol. 3:2441-249 (1983); Stoker and Bissell, J. Virol. 62:1008-1015 (1988)). One of these systems (Watanabe and Temin, supra) has been derived from the reticuloendotheliosis virus type A (REV-A). Sevoian et al., Avian Dis. 8:336-347 (1964). Replication-defective retroviral vectors derived from the REV-A virus are based on the helper cell line C3 (Watanabe and Temin, supra) which contains a packaging defective helper provirus. The derivation of the C3 helper line and several replication-defective retroviral vectors have been described in detail in U.S. Pat. No. 4,650,764, which is hereby incorporated by reference in its entirety. Several other retroviral vectors have been described by Emerman and Temin, Cell 39:459-467 (1984), which is hereby incorporated by reference in its entirety.
Shuman and Shoffner (Poult. Sci. 65: 1437-44 (1986)) used a replication-defective retroviral vector constructed by Emerman and Temin, supra, containing a bacterial gene for neomycin resistance and a herpes virus thymidine kinase gene, to inject into developing ovarian follicles prior to ovulation. This system involves gene transfer by injection at the single cell (ova) stage before fertilization and cleavage. Of 27 chicks examined two were tentatively identified with DNA sequences that hybridized to radiolabeled vector probes. However, the vector sequences were not completely characterized nor was the presence of replication competent virus ruled out. No progeny from these chickens were available to test whether any vector sequence was passed through the germ cells.
Thus, there is limited success to date in methods of gene transfer in chickens using replication-defective retroviral vectors, in particular, defective REV vectors. To date, there is no report of successful transfer of replication-defective retroviral vector sequences into developing chick embryos without the use of replication competent REV helper virus. The art has not yet been provided with a method of gene transfer into chicken or other avian embryos which permits the stable integration of vector into somatic and/or germ cells of the embryo using a replication-defective retroviral vector without significant helper virus. The problem has been to discover a stage of early avian development at which pluripotent stem cells of the embryo are both susceptible to infection by the replication defective retrovirus and able to be reached with sufficient vector to permit effective delivery to such cells. Thus, it can be seen that a need has existed for a reliable and effective method of transferring a nucleic acid sequence into somatic and germ cells of chicken embryos using a retroviral vector that inserts the nucleic acid sequence in embryonic cells in an initial round of infection but cannot reinfect the cells after the initial round of infection. This is especially critical for introducing genes into chickens or other avian species intended for commercial use.